Manufacturers add menthol as a flavoring in many tobacco products and in electronic cigarettes. Some smokers who use menthol find it more difficult to quit. Most suggestions about the basis for this phenomenon assume that menthol affects the pharmacokinetics of nicotine, but this explanation has several unsatisfactory aspects. The project explores a novel and transformative idea that menthol actually acts within cells, to modify the biosynthesis, stoichiometry, endoplasmic reticulum (ER) exit, trafficking, or membrane insertion of nicotinic acetylcholine receptors (nAChRs). Preliminary evidence is consistent with the idea that menthol, at submicromolar concentrations, is a chemical chaperone for both ?4?2 and ?6?2?3 nAChRs. The project expands the present measurements and also extends the studies to ?4?2 and ?6?2?3 nAChRs. An important underlying principle is the chaperoning generalization, which states that experiments in isolated cell lines are quite relevant for chaperoning processes in the neurons and in brains. Aim 1 distinguishes between acute (10 min) and chronic (24 hour) effects of menthol on the plasma membrane, including dose-response relations and waveform analysis for ACh and nicotine. A chaperoning effect is quite unlikely in 10 min but quite possible in 24 h. Aim 2 provides the mechanism of chronic effects of submicromolar menthol, by examining the entire pathway from synthesis of nAChRs in the ER, exit from the ER, cycling between Golgi and ER, nAChR movements within cells, to eventual plasma membrane (PM) composition of nAChRs. The experiments will proceed by analogy with the presumably more specific pharmacological chaperone effects of submicromolar nicotine. The effects of nicotine are under extensive study, and the methods adapt themselves well to examine the hypothesis that menthol is a chemical chaperone. Nearly all the tools and techniques now exist in our laboratory, requiring no extensive development. As in Aim 1, we will study any interactions between nicotine and menthol concentrations. Aim 2a Describes effects on functional nAChRs at the PM. The experiments utilize dose-response relations and for ACh itself and nicotine, waveform analysis, and superecliptic pHluorin measurements of PM and ER localization. Also, a novel set of measurements on fluorescent receptors in isolated plasma membrane will provide precise PM stoichiometry and subunit order within the assembled pentamer. Aim 2b then continues to define several additional possible effects of menthol on the cell biology of nAChRs, as the nAChRs are processed within cells. Experiment 2b1 measures menthol-produced changes to total / ER nAChR levels, using SEP-tagged nAChRs. We will also employ selective biotinylation (intact vs permeabilized cells) to determine the ratio of PM to total nAChRs. Experiment 2b2 monitors changes in intracellular stoichiometry, using F?rster resonance energy transfer (FRET). Experiment 2b3 studies changes in ER exit sites (ERES), a general assay for increased flux out of the ER. This experiment will be conducted both with and without expressed nAChRs, to test the generality of the proposed molecular chaperone effect. Experiment 2b4 monitors changes in COPI interactions, an assay for the importance of cycling between Golgi and ER. Experiment 2b5 monitors changes in the dynamics of intracellular nAChRs, using total internal reflection microscopy (TIRFM) video analysis. The data may provide novel insights about the actions of menthol on nAChRs, showing the details, limits, or alternatives to the chemical chaperone mechanism. The data will also shed light on menthol effects on the cell biology of other membrane proteins. With the data from this project in hand, a future R01 project can perform new sets of experiments on the effects of chronic menthol in cultured neurons, in brain slices, in neuronal circuits, in animal models, and in behavioral assays.